Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5–7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.


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JC-1 is a membrane permeant dye used to monitor the mitochondria membrane potential. Upon treatment of HEK-293 and LS174T cells with respective LD 50 of CuAc for 12 h and 24 h, cells were incubated with 5 µM JC-1 dye (Thermo fisher Scientific, Waltham, MA, USA) for 30min at 37ºC. Then cells were analyzed under a fluorescence microscope (EVOS FL auto, Thermo fisher Scientific, UK) with an excitation wavelength of 488 nm and an emission range between 515/545 nm and 575/625 nm respectively.
Additionally, to analyse mitochondrial potential, a separate set of cells were trypsinized after LD 50 CuAc treatment at 12 h and 24 h and then incubated with 2 μM JC-1 for 15 min at 37°C, 5% CO 2 . Cells were then washed with PBS and analyzed on a CytoFLEX S flow cytometer (Beckman, California, USA). The data was analyzed using the FlowJo software (version 10.6.1).

Annexin V apoptosis assay by flow cytometry
After treatment of cells with respective LD 50 of CuAc at different time intervals, cells were trypsinized and washed with ice cold PBS and suspended in annexin binding buffer (Thermo fisher Scientific, Waltham, MA, USA). Annexin V-FITC (Thermo fisher Scientific, Waltham, MA, USA) at a final concentration of 2 µg/ml was added to the cells and incubated for 15 min under dark conditions. Prior to flow cytometry, 2 µg/ml 7-AAD (Thermo fisher Scientific, Waltham, MA, USA) was added and cells were analyzed using a CytoFLEXS flow cytometer. The data was analyzed using the FlowJo software (version 10.6.1).

Western blot assay for protein expression
Cells were harvested with or without respective treatment conditions and washed with ice-cold PBS. Then, cells were incubated for 45 min in an ice bath with RIPA lysis buffer (Thermo Scientific, USA) with agitation at regular intervals to isolate whole cell protein extract. The lysate was then collected by centrifuging at 13,000 rpm for 10 min to remove any cell debris. The protein concentration was analyzed using a BCA reagent (Merck, New Jersey, USA) against a known standard curve. The protein lysate was mixed with 2X Laemmle sample loading buffer (Thermo Scientific, USA) and heated for 5min at 90ºC. Gel electrophoresis was performed for 90 min at 120V on a 4 -12% precast polyacrylamide gel (Bio Rad, California, USA). The proteins were then transferred to a PVDF membrane and blocked with 5% (w/v) non-fat skimmed milk in TBST (Tris buffered saline with 0.1% Tween-20) for 1h. The membrane was further incubated with respective primary antibodies of anti-CEA (Thermo Scientific, USA), caspase 3 and 9, PARP and β-actin (Cell Signalling Technologies, Beverly, USA). Antibody dilution used was in range from 1:1000 -1:1500 in TBST, overnight at 4ºC. Next, the membrane was washed with TBST for 1h and then labeled with respective HRP tagged secondary antibody (Cell Signalling Technologies, Beverly, USA) at a 1:5000 ratio in TBST, for 2 h at room temperature. The protein bands on the membrane were visualized by adding Pierce™ ECL reagent and quantified using a chemi-doc instrument S-4 (Biorad, USA). Band intensities were measured from independent replicates using ImageJ (NIH, USA).

Drug release experiments
The drug release study was done using the reverse dialysis method. 1 10mg/ml c of Cbs-Cu-Af in 1X PBS was placed under stirring conditions at 37ºC. A Slide-A-Lyzer™ MINI dialysis tube of 2K MWCO (Thermo Scientific USA) was placed in the PBS solution to entrap the free CuAc released from the cubosome. At regular intervals,100µl of the samples were taken from the dialysis tube (same amount of 1X PBS replaced) and was used for analysis of copper content using ICP-OES ( similar to the report used by Nguyen et al. for detection of cisplatin). 2 Similarly, a known concentration of CuAc was stirred placed in a dialysis tube was used to check for release kinetics for free drug. Table S1. Cubosome synthesis and size distribution study by DLS. The hydrodynamic diameter of cubosomes and their polydispersity index (PDI) with different ratio of MO, DPA and pluronic F127 as stabilizer. Data shows 95: 5 w/w % of MO:DPA and 7% (w/w) F127 produced the smallest size (106 nm) of bare cubosomes and thus this composition was selected for further studies.   Figure S1: Chemical characterization of cubosome upon functionalization and drug loading. Energy Dispersive X-Ray analysis (EDAX) of (A) bare Cbs and (B) CuAc encapsulated cubosomes tagged with Affimer (Cbs-Cu-Af). The latter showing distinct peaks for Copper (black arrows) which confirms the presence of CuAc in the cubosome. The characteristic Sulphur Kα and Kβ peaks (2.3 -2.5 eV) arising from the tagged cubosomes are also seen here (red arrows) that is due to cysteine present in the Affimer (C) Fourier-transform infrared spectra of Affimer tagged (Cbs-Cu-Af) and untagged (Cbs-Cu) cubosomes loaded with CuAc show that Cbs-Cu shows a peak at 2127 cm -1 attributed to the azide (N=N=N) functional group . After conjugation of DBCO Affimer to the DSPE-azide group on the cubosomes, the peak is absent in the spectrum for Cbs-Cu-Af which confirmes the covalent linkage of Affimer to the cubosomesurface and is not just physical adsoprtion. (D) Drug release data as determined by ICPOES concluded that CuAc has a slow and sustained release from the cubosomes.

Affimer proteins as promising ligands for carcinoembryonic antigen on colorectal cancer cells
CEA binding Affimer proteins were identified using a 'phage display library method' by Shamsuddin et al. 3 . Out of the three CEA binding Affimers identified, clone II and III were chosen for this study having 9 and 10 distinct amino acid residues at the variable region in clone II and clone III respectively. Based on a 50 ml of working volume, the yield of the Affimers was 8.3 mg and 6.27 mg for clone II and III respectively 3-4 with corresponding molecular weights noted to be 12.5 and 12.6 kDa ( Figure S3A). The surface plasmon resonance showed that the Affimers demonstrated a high binding affinity towards CEA (K d for clone II:15.3 ± 0.37 nM; Kd for clone III: 34.4 ± 16 nM) ( Figure S3B). Interestingly when compared to CEA mAb, these Affimers showed enhanced binding to cell surface CEA as studied from immunofloresence assay on CRC cells.       S-20 Figure S14: 3D reconstruction of tumor volume from ultra sonography measuredat day 11 and day 13 in the three groups of mice. The Cbs-Cu-Af (Affimer targeted) administered group showed restricted tumor growth on day 13 compared to the rest of the two groups.
S-21 Figure S15: Off-target toxicity study in the three groups of mice by haematoxylin and eosin staining (H&E) of kidney, liver and heart. As evident from the images there is no sign of toxicity or tissue necrosis arising from administration of Cbs-Cu (non-targeted) or Cbs-Cu-Af (Affimer targeted) cubosomes.

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Figure S16: Off-target toxicity study in the three groups of mice by tissue examination of lung, brain and spleen by haematoxylin and eosin staining (H&E). As evident from the images there is no sign of toxicity or tissue necrosis arising from administration of Cbs-Cu (non-targeted) or Cbs-Cu-Af (Affimer targeted) cubosomes.